The disulphide bond catalyst DsbA, a key component of the disulphide bond (Dsb) forming machinery, is a central mediator of bacterial pathogenesis, as it promotes the assembly of a wide range of virulence factors required at different stages of the infection process. Even though extensive research has been focused in deciphering the Dsb systems in pathogens, to date, little is known about the way Dsb proteins interact with and fold virulence substrates. Interestingly, the main causative agent of urinary tract infections, Uropathogenic E. coli (UPEC), possesses two DsbA enzymes, namely DsbA and DsbL, for virulence factor folding. Through a combination of molecular biology, structural biology and biophysics, this work is focussed on understanding the structural and molecular determinants behind the substrate interaction and specificity of these two distinct DsbA homologues. Outcomes of this research will elucidate how bacterial pathogens use the Dsb system to generate virulence proteins involved in the establishment of infection. This knowledge will allow us to understand how bacteria cause disease and will provide important information to current campaigns aiming at developing DsbA inhibitors as potential anti-virulence agents.